The goal of this project is to develop effective molecularly-defined immune adjuvants for active immunization[unreadable] against cancer. Although tolerance to cancer is reversible, immunization against cancer antigens faces[unreadable] substantial hurdles related to tolerance and immune regulation. Potent immune adjuvants will be necessary[unreadable] for successful active immunization against cancer antigens, which are inherently poorly immunogenic. We[unreadable] apply recent knowledge about molecules that regulate growth, activation and survival of innate and acquired[unreadable] immune cells to study adjuvant strategies and underlying mechanisms. We use genetic adjuvants (plasmid[unreadable] DNA incorporating Fc domains of IgG) combined with DNA vaccines against cancer antigens. DNA[unreadable] encoding cytokines have been carefully selected for analysis based on results of extensive preliminary[unreadable] screening and on our understanding about how these molecules regulate activation and survival of innate[unreadable] and acquired immune cells. Specific aim 1 is built on experimental results from our screens showing that[unreadable] DNA encoding an IL-12/Fc fusion molecule is most effective at enhancing T cell responses and tumor[unreadable] immunity. A phase I clinical trial is proposed to assess safety of IL-12/Fc DNA and compare the relative[unreadable] immunologic efficacy of vaccination with tyrosinase DNA plus IL-12/Fc DNA adjuvant to tyrosinase DNA[unreadable] alone in patients with stage III melanoma. Specific aim 2 examines why ligation of Fc domains to cytokines[unreadable] markedly increases adjuvanticity while unexpectedly decreasing their bioavailability. Genetic approaches[unreadable] are used to address the hypothesis that adjuvant effects are amplified by interactions with Fc receptors on[unreadable] innate immune cells. Specific aim 3 studies DNA adjuvants combined with vaccination in treatment models[unreadable] of melanoma, including treatment in a transgenic model that develops de novo melanoma. Subaims[unreadable] investigate a) the role of IFNgamma produced by NK cells for adjuvanticity by cytokine and cytokine/Fc cDNAs,[unreadable] and b) adjuvant effects of IL-23 DNA, which is strongly implicated in the pathogenesis of inflammatory[unreadable] autoimmune diseases. Specific aim 4 investigates the role of cytokine DNA adjuvants for potentiating[unreadable] antigen-specific responses elicited by vaccination during homeostatic recovery of the immune system after[unreadable] non-myeloablative treatments. The overall goal is to develop the most potent DNA adjuvants that can be[unreadable] applied to vaccines delivered during homeostatic recovery.[unreadable]